This invention relates to the viral enterotoxin NSP4 and to methods for using it, or antibodies/antisera thereto, as diagnostic agents, vaccines and therapeutic agents for the detection, prevention and/or treatment of rotaviral disease, for the prevention of stunted growth in animals and children caused by rotaviral infection and for the treatment of cystic fibrosis. This invention also relates to methods and animal models for 1) the screening for viral enterotoxins, 2) the detection of viral enterotoxins and 3) the identification of viral enterotoxins.
Rotaviruses are the leading cause of severe, life-threatening viral gastroenteritis in infants and animals (Kapikian et al., 1996) and are associated with sporadic outbreaks of diarrhea in elderly (Halvorsrud 1980) and immunocompromised patients (Holzel et al., 1980). These viruses have a limited tissue tropism, with infection primarily being restricted to cells of the small intestine (Estes et al., 1994). Rotavirus infections also cause morbidity and mortality in many animal species. Moreover, the outcome of infection is age-related; although rotaviruses may infect individuals and animals of all ages, symptomatic infection (i.e., diarrhea) generally occurs in the young (6 monthsxe2x80x942 years in children, and up to 14 days in mice), and the elderly.
Age-related host factors which may influence the outcome of infection have been proposed to include 1) differences in the presence/quantity of virus-binding receptors on mature villus epithelial cells, 2) virus strains with a specific spike protein (VP4), 3) passive immunity acquired by maternal antibody or in colostrum, and 4) reduced levels of proteases in the young.
Disease resulting from rotavirus infection in mice has been studied more extensively than in any other species and an age restriction of disease has been reported by several investigators (Ramig 1988; Wolf et al. 1981; Riepenhoff-Talty et al., 1982). Only mice less than 14 days of age develop diarrhea following oral inoculation of murine rotavirus, and the peak age at which animals are most likely to develop diarrhea (6-11 days) corresponds to the age when rotavirus can bind to mouse enterocytes (Riepenhoff-Talty et al., 1982). Treatment of 8 day old mice with cortisone acetate which promotes premature maturation of intestinal epithelial cells, results in a reduced susceptibility to rotavirus-induced diarrhea, although the mice can still be infected (Wolf et al., 1981). These data were interpreted to suggest that the capacity of murine rotaviruses to induce diarrhea in young, but not adult mice, is due to the quantity of rotavirus-binding receptors on the surface of villus epithelial cells in the young mouse intestine.
When compared to rotavirus infections in other species, rotavirus infections in mice show minimal histologic alterations. That is, villus blunting is limited and transient, and crypt cell hyperplasia is not present. In addition, the loss of villus tip epithelial cells is more limited in mice than in other animals. Instead, vacuolization of enterocytes on the villus tips is a predominant feature in symptomatic rotavirus infection in mice and virus replication may be abortive (Greenberg et al., 1981). The lack of extensive pathologic alterations in the mouse intestine during symptomatic infections has remained a puzzle; one interpretation of this phenomenon is that a previously unrecognized mechanism of diarrhea induction may be active in symptomatic rotavirus infection in mice.
Despite the prevalence of rotavirus infections and extensive studies in several animal models and many advances in understanding rotavirus immunity, epidemiology, replication and expression, rotavirus pathogenesis, specifically, the mechanism of diarrhea induction, remains poorly understood. Proposed pathophysiologic mechanisms by which rotaviruses induce diarrhea following viral replication and viral structural protein synthesis include malabsorption secondary to the destruction of enterocytes (Graham et al., 1984), disruption of transepithelial ion homeostasis resulting in fluid secretion (Collins et al., 1988), and local villus ischemia leading to vascular damage and diarrhea (Osborne et al., 1988). However, these proposed mechanisms do not explain cases of rotavirus-induced diarrhea observed prior to, or in the absence of, histopathologic changes (Theil et al., 1978; McAdaragh 1980; Saif et al., 1976).
On the other hand, the pathophysiology of bacterial-induced diarrhea based on interactions with intestinal receptors and bacterial enterotoxins is well understood (Burges et al., 1978; Gianella et al., 1981; Krause et al., 1990). The heat-stable toxin A and the heat-labile toxin of E. coli, and guanylin (an endogenous, 15 amino acid intestinal ligand originally isolated from rat jejunum) induce diarrhea by binding a specific intestinal receptor, increasing cAMP or cGMP, and activating a cyclic nucleotide signal transduction pathway (Giannella et al., 1993; Currie et al., 1992; Field et al., 1978; Forte et al., 1992). The net effect of these bacterial toxins is to increase Clxe2x88x92 secretion, and decrease Na+ and water absorption.
Previous studies in insect cells indicated that a receptor-mediated phospholipase C pathway is associated with the increases in [Ca2+]i, following exogenous treatment of cells with NSP4 or NSP4 114-135 peptide (Tian et al., 1994). The rotavirus nonstructural ER glycoprotein, NSP4, has been shown to have multiple functions including the release of calcium from the endoplasmic reticulum (ER) in SF9 insect cells infected with recombinant baculovirus containing the NSP4 cDNA (Tian et al., 1994; Tian et al., 1995). In addition, NSP4 disrupts ER membranes and may play an important role in the removal of the transient envelope from budding particles during viral morphogenesis. NSP4 114-135, a 22 aa peptide of NSP4 protein, has been shown to be capable of mimicking properties associated with NSP4 including being able to (i) mobilize intracellular calcium levels in insect cells when expressed endogenously or added to cells exogenously (Tian et al., 1994; Tian et al., 1995), and (ii) destabilize liposomes.
Expression of NSP4 in insect cells increased [Ca2+]i levels from a subset of the thapsigargin-sensitive store (ER) (Tian et al., 1995). The [Ca2+]i mobilized by NSP4 or the NSP4 114-135 peptide added exogenously to cells was blocked by a phospholipase C inhibitor, the U-73122 compound, suggesting that a receptor-mediated pathway is responsible for the calcium release from the ER induced by NSP4 (Tian et al., 1995). The [Ca2+]i mobilized by NSP4 expressed intracellularly was not blocked by the U-73122 compound, suggesting that a second pathway is responsible for the calcium release from the ER induced by intracellular NSP4 (Tian et al., 1995).
The present invention discloses herein a method of immunization against rotavirus infection or disease comprising administering to a subject a peptide NSP4 112-175 or NSP4 112-150 or a toxoid thereof. Further, the present invention discloses a method of immunization against rotavirus infection or disease comprising administering to a subject a non-gylcosylated NSP4 protein or a toxoid of NSP4. The immunizations may result in both homotypic and heterotypic immunity.
In another specific embodiment, it is also provided a method of passive immunization against rotavirus infection comprising administering to an expectant mother a peptide NSP4 112-175, NSP4 112-150 or a toxoid thereof. Yet further, the present invention discloses a method of passive immunization against rotavirus infection comprising administering to an expectant mother a non-gylcosylated NSP4 protein or a toxoid of NSP4. The immunizations may result in both homotypic and heterotypic immunity.
A specific embodiment of the present invention is that the NSP4 peptide (e.g., NSP4 112-175 or NSP4 112-150) or toxoid is produced by a synthetic method. In a further specific embodiment, the NSP4 peptide or toxoid is produced by an expression vector. The expression vector is selected from the group consisting of mammalian, yeast, bacterial or insect.
Another embodiment of the present invention is a fusion protein comprising a protein that forms a virus-like particle linked to a NSP4 peptide. The fusion protein further comprises a linker sequence. An exemplary linker sequence includes, but is not limited to, three alanine residues and an alanine and serine residue. It is also contemplated that three glycine residues may be substituted for the three alanine residues. One of skill in the art is cognizant that the scope of the invention is not limited to a five residue linker. It is contemplated that other linkers may be used, for example, but not limited to, three alanine residues or 3 glycine residues. The NSP4 peptide is NSP4 112-175 or a toxoid thereof or NSP4 112-150 or a toxoid thereof. The protein that forms a virus-like particle is a viral protein or peptide isolated from the viral families Caliciviridae or Reoviridae.
In specific embodiments, the viral protein isolated from Caliciviridae is a Norwalk virus protein or peptide. In two particular fusion proteins, the Norwalk virus protein is ORF2 or ORF2 plus ORF3 or a fragment or toxoid of ORF2 or ORF2 plus ORF3. Specifically, ORF2 comprises amino acids 21-530 and ORF3 comprises 1-212.
In yet another specific embodiment, the viral protein or peptide isolated from Reoviridae is a rotavirus protein or peptide. More particularly, the rotavirus peptide is selected from the group of rotavirus proteins consisting of VP2, VP4, VP6 and VP7. In specific embodiments, the rotavirus peptide is VP2 or a VP2 fragment. Specifically VP2 comprises amino acids 94-881.
Another embodiment of the present invention discloses an expression vector comprising a nucleic acid sequence encoding a fusion protein operatively linked to a first promoter sequence, and a nucleic acid sequence encoding a viral peptide that is part of a virus-like particle operatively linked to a second promoter sequence. The fusion protein comprises a viral peptide that is part of a virus-like particle linked to a NSP4 peptide. The viral peptide linked to the NSP4 peptide is VP2 and forms the inner shell of the virus-like partilce. The viral peptide that is not linked to the fusion protein is rotavirus VP6. This peptide forms the outer shell of the virus-like particle surrounding the VP2 shell. The first promoter sequence is a polyhedrin promoter sequence and the second promoter sequence is a p10 promoter sequence. The expression vector is selected from the group consisting of insect, mammalian, viral and bacterial
In specific embodiments, the expression vector comprises a fusion protein that comprises a nucleic acid sequence encoding rotavirus VP2 amino acids 94-881 linked to a nucleic acid sequence encoding NSP4 amino acids 112-175.
In another embodiment, the expression vector comprises a fusion protein that comprises a nucleic acid sequence encoding rotavirus VP2 amino acids 94-881 linked to a nucleic acid sequence encoding NSP4 amino acids 112-150.
Another embodiment of the present invention comprises an expression vector comprising a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises a viral peptide that is part of a virus-like particle linked to a NSP4 peptide. The nucleic acid sequence is operatively linked to a promoter sequence.
In specific embodiments, the expression vector comprises the fusion protein that comprises a nucleic acid sequence encoding Norwalk virus ORF2 linked to a nucleic acid sequence encoding NSP4 amino acids 112-175. In a further embodiment, the fusion protein comprises a nucleic acid sequence encoding Norwalk virus ORF2 linked to a nucleic acid sequence encoding NSP4 amino acids 112-150. It is also contemplated that the fusion protein may be a fragment of Norwalk virus ORF2 linked to NSP4 amino acids 112-175 or a fragment of Norwalk virus ORF2 linked to NSP4 amino acids 112-150.
In another embodiment, the expression vector comprises the fusion protein that comprises a nucleic acid sequence encoding a nucleic acid sequence encoding Norwalk virus ORF2 and ORF3 linked to NSP4 amino acids 112-175. In another embodiment the fusion protein comprises a nucleic acid sequence encoding Norwalk virus ORF2 and ORF3 linked to a nucleic acid sequence encoding NSP4 amino acids 112-150. It is also contemplated that the fusion protein may be a fragment of Norwalk virus ORF2 and ORF3 linked to NSP4 amino acids 112-175 or a fragment of Norwalk virus ORF2 and ORF3 linked to NSP4 amino acids 112-150.
A specific embodiment of the present invention also comprises a vaccine for inducing the formation of protective antibodies against rotavirus infection comprising administering a chimeric virus-like particle. The chimeric virus-like particle comprises a peptide NSP4 112-175, a first viral protein that is part of a virus-like particle, and a second viral protein that is part of a virus-like particle. Specifically, the first viral protein is rotavirus VP2, which forms an inner shell and said second viral protein is rotavirus VP6, which forms an outer shell surrounding the VP2 shell.
In another embodiment, it is also provided a method of immunization against rotavirus infection or disease comprising the step of administering to a subject a compound comprising a chimeric virus-like particle. The chimeric virus-like particle comprises a peptide NSP4 112-175, a first viral protein that is part of a virus-like particle, and a second viral protein that is part of a virus-like particle. More particularly, the first viral protein is rotavirus VP2 and said second viral protein is rotavirus VP6. The compound is administered orally, parenterally or intranasally. Another aspect comprises that the compound is administered with an adjuvant.
In specific embodiments, the compound is simultaneously or consecutively administered by at least two different routes of administration. Exemplary routes of administration include, but are not limited to, oral, parenteral or intranasal.
Another embodiment of the present invention comprises a method of inducing an immune response comprising the step of administering to a mammal one expression vector, wherein said expression vector comprises a nucleic acid sequence encoding a fusion protein, wherein said fusion protein comprises a first viral protein that is part of a virus-like particle linked to a NSP4 nucleic acid sequence, and a nucleic acid sequence encoding a second viral protein that is part of a virus-like particle. In specific embodiments, the nucleic acid sequence encoding the fusion protein and the nucleic acid sequence encoding the second viral protein are under separate transcriptional control and wherein the nucleic acid sequence encoding the fusion protein and the nucleic acid sequence encoding the second viral protein are in tandem in the one expression vector.
A specific embodiment also provides a method of inducing an immune response comprising the steps of co-administering to a mammal or a cell two different expression vectors, wherein a first expression vector comprises a nucleic acid sequence encoding a first viral protein that is part of a virus-like particle and a second expression vector comprises a nucleic acid sequence encoding a fusion protein, wherein said fusion protein comprises a second viral protein that is part of a virus-like particle linked to a NSP4 nucleic acid sequence.
Other embodiments, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.